In 1985, Smith first demonstrated that filamentous phages tolerate foreign protein fragments inserted in their gene III protein (pIII), and could show that the protein fragments are presented on the phage surface (Smith, 1985). Ladner extended this concept to the screening of repertoires of (poly)peptides and/or proteins displayed on the surface of phage particles (WO 88/06630; WO 90/02809). Since then, phage display has experienced a dramatic progress and resulted in substantial achievements.
Various formats have been developed to construct and screen (poly)peptide/protein phage-display libraries, and a large number of review articles and monographs cover and summarise these developments (e.g., Kay et al., 1996; Dunn, 1996; McGregor, 1996). Most often, filamentous phage-based systems have been used.
Initially proposed as display of single-chain Fv (scFv) fragments (WO 88/06630; see also WO 92/01047), the method has rapidly been expanded to the display of other (poly)peptides/proteins, such as bovine pancreatic trypsin inhibitor (BPTI) (WO 90/02809), peptide libraries (WO 91/19818), human growth hormone (WO 92/09690), and various other proteins, including the display of multimeric proteins such as Fab fragments (WO 91/17271; WO 92/01047).
To anchor the (poly)peptides/proteins to the filamentous bacteriophage surface, mostly genetic fusions to phage coat proteins are employed. Preferred are fusions to gene III protein (Parmley & Smith, 1988) or fragments thereof (Bass et al., 1990), and gene VIII protein (Greenwood et al., 1991). In one case, gene VI has been used (Jespers et al., 1995), and in one case, a combination of gene VII and gene IX has been used for the display of Fv fragments (Gao et al., 1999).
Furthermore, phage display has also been achieved on phage lambda. In that case, gene V protein (Maruyama et al., 1994), gene J protein, and gene D protein (Sternberg & Hoess, 1995; Mikawa et al., 1996) have been used.
Besides using genetic fusions, foreign peptides or proteins have been attached to phage surfaces via association domains. In WO 91/17271, it was suggested to use a tag displayed on phage and a tag-binding ligand fused to the (poly)peptide/protein to be displayed to achieve a non-covalent display.
A similar concept was pursued for the display of cDNA libraries (Crameri & Suter, 1993). There, the jun/fos interaction was used to mediate the display of cDNA fragments. In their construct, additional cysteine residues flanking both ends of jun as well as fos further stabilised the interaction by forming two disulfide bonds.
One question used to be, how to best recover phages which have bound to the desired target. Normally, this is achieved by elution with appropriate buffers, either by using a pH- or salt gradient, or by specific elution using soluble target. However, the most interesting binders which bind with high affinity to the target might be lost by that approach. Several alternative methods have been described which try to overcome that problem, either by providing a cleavage signal between the (poly)peptide/protein being displayed and its fusion partner, or between the target of interest and its carrier which anchors the target to a solid surface. Furthermore, most of the approaches referred to hereinabove require the use of fusion proteins comprising at least part of a phage coat protein and a foreign (poly)peptide/protein.
In WO 01/05909, an entirely different system is described which does not require fusion proteins, and hence solved many of these problems. The so-called “CysDisplay” system, described in WO 01/05909, is based on the formation of a covalent disulphide bond between a bacteriophage coat protein and an immunoglobulin or a functional fragment thereof. The immunoglobulin, or the functional fragment thereof, is displayed on the surface of a bacteriophage particle. A similar technology was subsequently disclosed in WO 03/060065. WO 03/060065 differs from WO 01/05909 in that the Cys-tagged pIII polypeptide is provided via a modified helper phage rather than a phagemid. Furthermore, WO 03/060065 also mentions other adapters that might be employed to display (poly)peptides/proteins on bacteriophage particles, such as homomultimeric proteins (PDGF, Max, RelA, neurotrophin) and heteromultimeric proteins (proteink kinase complexes, SH2-domain conating proteins, a-Pal/Max, Hox/Pbx).
Although the CysDisplay system is functioning well, a system which displays higher amounts of the (poly)peptides/proteins on the bacteriophage particles can be advantageous in certain situations, for example, such an improved system with increased display rates, in particular an increased functional display rate, would surely be beneficial and enable the more convenient, reliable and specific isolation of binders, in particular binders which bind to their target with high affinity.
Snyder et al., 1981, investigated the local environment of reactive cysteine residues of peptides of naturally occurring proteins by treatment with 2-nitrobenzoic acid and observed that cysteine residues surrounded by positively charged amino acids showed higher reactivity. In a follow up study Snyder et al., 1983, investigated the kinetics of disulfide formation and came to essentially the same conclusions. Bulaj et al., 1998, investigated the kinetics of 16 model peptides for their capability to form disulphide bond with various non-proteinaceous molecules. They observed that the presence of net charges on the peptides and on the non-proteinaceous reagents have influence on the reactivity. Britto et al., 2002, investigated the electrostatic environment of intramolecular disulphide bonds in tubulin and found that the most reactive cysteine residues were within 6.5 Angstrom of positively charged residues, presumably promoting dissociation of the thiol to the thiolate anion. Hansen et al., 2005, investigated intramolecular disulphide bonds of engineered YFP and found an increase in reactivity if positively charged amino acids are present in the proximity of the reactive cysteine residues. Albrecht et al., 2006, reported the generation of monospecific multivalent Fab's attached to PEG via cysteine residues.
However, none of the studies above describe a system in which an intermolecular disulphide bond is formed between a first (poly)peptide/protein and a second, different, (poly)peptide/protein. In particular, in none of these studies is such a disulphide bond formed in the periplasmatic space of a host cell. Furthermore, in none of the cited studies is a (poly)peptide/protein displayed on the surface of a bacteriophage particle.